This proposal targets the molecular basis of iron overload disorders and iron-restricted anemia, which are among the most common hematological diseases worldwide. Recent studies have documented the iron-regulatory hormone hepcidin as the key molecule responsible for systemic iron homeostasis. Hepcidin expression is mediated via the bone morphogenetic protein signaling pathway and requires the involvement of other key plasma membrane proteins, including hemojuvelin (HJV), hemochromatosis protein (HFE), transferrin receptor-2 (TfR2), and neogenin. Mutations in the HJV, HFE, or TfR2 genes in humans reduce hepcidin expression and cause hereditary hemochromatosis. Conversely, matriptase-2 (MT2) is a robust suppressor for hepcidin expression. Mutations in the MT2 gene in humans result in increased hepcidin expression, which leads to iron-refractory iron-deficiency anemia. However, the precise mechanisms by which neogenin induces, MT2 suppresses, and iron modulates hepcidin expression are poorly understood. Our long- term goal is to understand the mechanism of systemic iron homeostasis. The objective of this grant is to characterize the coordination of neogenin, MT2, and the hepatocyte growth factor activator inhibitor-2 (HAI-2) in the regulation of hepcidin expression. Our central hypothesis is that neogenin acts as a scaffold to facilitate hepcidin expression in hepatocytes, and that bodily iron load negatively regulates MT2 activity through HAI-2 to adjust hepcidin expression to an appropriate level. This hypothesis has been formulated on the basis of the data produced by the applicants? and other laboratories. The rationale for the proposed research is that understanding the regulation of hepcidin expression by iron has the potential to develop new therapies for iron overload disorders and iron-restricted anemia. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the underlying mechanism by which hepatic neogenin facilitates hepcidin expression. 2) Test the hypothesis that MT2 coordinates with HAI-2 to modulate hepcidin expression in response to bodily iron load. The approach is innovative, because it focuses on the mechanistic studies of neogenin, MT2, and HAI-2 in iron-regulated hepcidin expression at molecular, cellular, and systemic levels. The proposed research is significant, because it is expected to provide the basis for the development of pharmacologic strategies. Successful completion of these studies will not only increase our understanding of systemic iron homeostatic mechanism but also lay the foundation for translating these advances into tangible benefits for patients with iron disorders.